Mutant TP53 switches therapeutic vulnerability during gastric cancer progression within interleukin-6 family cytokines

Summary Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in the glandular epithelium of KrasG12D;Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047R is sufficient to induce adenomas and that lesions progress to carcinoma when also harboring Pten deletions. An additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with the poorer survival of patients with high IL-6 expression, we identify IL-6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.


Figure S1 . 1 AC
Figure S1.Frequency of KRAS, PI3K and TP53 pathway alterations in human gastric cancer and PI3k pathways potency as cancer driver in mice, Related to Figure 1 A Top 10 mutated (in percent) oncogenic pathways in stomach adenocarcinoma (STAD) patients.Data extracted from TCGA data set (STAD n=383).RTK/RAS= Receptor tyrosine kinase/RAS.B Table shows selected pathways mutation frequencies (as in A) for single, double and triple pathway involvement.C Venn diagram for oncogenic signaling pathway activation gene signature analysis, showing number of patients with STAD with KRAS and/or PI3K pathway activation (based on gene expression signature) and TP53 gene mutation (silent mutation (n=1) excluded).63 patients had neither TP53 mutation nor KRAS or PI3K activation status tumors (STAD n=414).D Data from C presented in table format, showing the precent frequency of patients with single, double, and triple pathways activation (*TP53 data is gene mutation based).E Representative whole-mount stomachs from Cre-positive mice of the indicated genotypes, collected at 6-18 months (as indicated) after Tamoxifen administration (arrowhead and black lines indicate tumors).F,G H&E-stained cross-sections, cut along the dotted line (in D), extend from the forestomach to the proximal end of the small intestine (SI) (E).Boxed regions are shown in higher magnifications (F).Scale bars 100µm.H Frequency (in percent) of tumor development in Cre-positive mice of the indicated genotype.

Figure S2 .
Figure S2.Nuclear β-Catenin and WNT-gene signature analysis in tumors without overt canonical WNT signaling pathway mutations A Representative microscopy images of anti-β-catenin IHC stained tumors of KPP, KPT and Apc KO (Tff1 CreERT2 ;Apc fl/fl ) mice.KPP and KPT image represent strongest staining (of n=10 mice analyzed).Apc KO shown as positive control for nuclear β-catenin positivity; Apc KO mice develop non-invasive adenomas.Scale bars equal 300 µm (top images) and 100 µm (bottom images).

Figure S3 .A
Figure S3.TP53 mutations status at genetic, protein and phenotype level, Related to Figure 2 A Upon tamoxifen administration Tff1 CreERT2 recombinase drives removal of the Los-Stop-Lox (LSL) cassette from the LSL-R172H allele to yield the R172H mutant expression, which is classified a Gain-of-Function (GoF) mutant.Some cells do not undergo Cre-recombination and retain the unrecombined LSL-R172H allele, which is a Loss-of-Expression (LoE) mutant (or "null' allele) as no or minimal TP53 protein

Figure S4 . 3 A
Figure S4.IL6 and IL11 expression profiles in mouse KPT tumor organoids and human GC cells, Related to Figure 3

Figure S6 .AB
Figure S6.STAT3 signaling quantification and IL6 cytokine expression on human gastric cancer Tissue Micro Arrays, Related to Figure 5 A Halo analysis of pSTAT3 stained human GC Tumor Micro array (TMA).Left image is the original pSTAT3 image of one representative tumor core of the TMA.Middle image shows the tissue segmentation: halo

Figure S7 .
Figure S7.Graphical summary of TP53 mutation driven IL-6 family cytokine dependency switch Illustrates the pathway activation series with corresponding gastric tumor progression, associated IL11, IL6 expression, STAT3 signalling activity and resulting cytokine dependencies.n/a = not applicable, nd = not determined, intermed.= intermediate.This figure was created with Biorender.com.